We have discovered the first-ever inhibitors of PF4, a platelet protein central to the pathophysiology of heparin induced thrombocytopenia (HIT). Heparin is a naturally-occurring anticoagulant that prevents the formation of clots and extension of existing clots within the vasculature, and major medical applications of heparin include dialysis, cardiac catheterization, and cardiopulmonary bypass surgery. Heparin therapy is usually safe and effective; however some patients develop HIT as a serious complication caused by an immunological reaction that targets platelets leading to a low platelet count (thrombocytopenia). HIT increases the risk of blood clots forming within blood vessels and blocking the flow of blood (thrombosis), referred to as HITT when thrombosis occurs. HITT develops in approximately 1-3% of patients treated with heparin for 5-10 days. Affected individuals have a 20-50% risk of developing new thromboembolic events, a mortality rate ~20%, and an additional ~10% of patients require amputations or suffer other major morbidity. Current treatment for HIT relies on elimination of heparin exposure from patients with suspected HIT and administration of direct thrombin inhibitors, which carry a significant risk of bleeding. Despite the removal of heparin from these patients, they remain at significant risk for thrombosis and death. We have discovered a novel approach to the treatment of HIT via the destabilization of the functionally-active PF4 tetramers to inactive monomers and dimers. The tetrameric form of PF4 binds to heparin to form Ultra-large Complexes (ULC). We have recently reported on the identification and characterization of PF4 antagonists (PF4As) that disrupt PF4 tetramerization, which is a prerequisite for ULC formation, and inhibit the formation of ULCs. In this grant proposal further we optimize our current series of PF4 tetramerization inhibitors (lead compound FC-3157) by designing and preparing new analogs that have improved potency and similar or better ADMET and drug-like properties. Using the combined expertise of Fox Chase Chemical Diversity Center, Inc. in medicinal chemistry and drug discovery, and the Sachais Laboratory at the New York Blood Center in HIT-based approaches, we anticipate this Phase II project to result one or more PF4 lead antagonists suitable for IND safety evaluation and ultimate evaluation in a clinical setting under IV administration. The first aim is lead optimization synthetic and medicinal chemistry. Aim 2 is the evaluation of analogs in in vitro assays for targeted activities (inhibition of PF4 tetramerization, ULC inhibition, and inhibition of cellular activation). Aim 3 is for the evaluation of analogs for acceptable ADMET drug values including: metabolic stability (microsomes), aqueous solubility, lipophilicity, CypP450 inhibition (7 isozymes most likely to cause exposure variability and drug-drug interactions), and in vivo pharmacokinetics in mice after IV administration. Safety assays on selected top compounds include 1) in vivo tolerability (MTD) in mice, 2) hERG and 3) Ames evaluation. Aim 4 involves evaluation of select analogs in the mouse HIT model.